We have posed the hypothesis that certain actions of insulin, which are known to occur independently of altered cAMP levels, may be mediated by insulin induced, cyclic AMP independent, alterations in protein phosphorylation. We have identified, in adipose tissue and liver, peptides whose phosphorylation is stimulated by insulin without concomitant alterations in cAMP content, or in the activity of the cAMP dependent protein kinase. The identity of these peptides is currently unknown. Our goal is to isolate one or more of these peptides, identify it, and use it as a substrate to study further the enzymes mediating its phosphorylation and dephosphorylation. The regulation of these protein kinases and protein phosphotases by insulin will be examined. Special emphasis is placed on the search for regulators of the activity of such enzymes which are generated in response to insulin. Th aim of these studies is to reconstitute in vitro, from purified components, the system mediating insulin. This work will provide new insights into the mechanism of insulin action.